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Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates.

Froissard M, Belgareh-Touzé N, Dias M, Buisson N, Camadro JM, Haguenauer-Tsapis R, Lesuisse E - Traffic (2007)

Bottom Line: Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells.Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments.Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Trafic intracellulaire des protéines dans la levure, Département de biologie Cellulaire, Institut Jacques Monod, Unité Mixte de Recherche 7592 CNRS-Universités Paris 6 et 7, France.

ABSTRACT
We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

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The AF-FOB conjugate does not reach its target in vivo.A) The IC50 values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. 55Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of 55Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.
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fig09: The AF-FOB conjugate does not reach its target in vivo.A) The IC50 values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. 55Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of 55Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.

Mentions: Acifluorfen is a powerful inhibitor of protoporphyrinogen oxidase, a mitochondrial enzyme catalysing the formation of protoporphyrin IX from protoporphyrinogen in the heme biosynthetic pathway. We covalently coupled this inhibitor to DFOB, using the same chemical coupling procedure as for Ga-DFOB-NBD (Methods) and assessed the inhibitory effect of (D)FOB-AF with respect to AF on protoporphyrinogen oxidase activity in vitro. The siderophore drug conjugate displayed weaker inhibitory activity than AF alone (Figure 9A), particularly when iron was bound to the siderophore moiety. This was probably because of steric constraints, as the substrate-binding domain (which is also the inhibitor-binding domain) of protoporphyrinogen oxidase encompasses a narrow active site cavity (37). Despite this limitation, the IC50 values measured with DFOB-AF and FOB-AF were nonetheless low enough (about 10−7 m and 10−6 m, respectively; Figure 9A) for the drug conjugates to be used as potent inhibitors of heme synthesis in yeast. Nevertheless, although AF-methyl efficiently inhibited S. cerevisiae growth on agar plates, the siderophore drug conjugate did not (Figure 9B). We then checked whether the cells took up FOB-AF efficiently. Both the fluorescent analog of FOB (FOB-NBD) and the siderophore drug conjugate (FOB-AF) were taken up by the cells through Sit1, although less efficiently than FOB itself (Figure 9C). We cultured cells bearing the SIT1 gene under control of the GAL1 promoter with galactose as the carbon source and in the presence of FOB-AF or FOB (100 μm). The washed pellets of cells cultured overnight in these conditions were brownish yellow in colour (the colour of undissociated siderophore), indicating that they had accumulated large amounts of FOB/FOB-AF (data not shown). The cells grown with FOB-AF showed no growth defect (and no porphyrin accumulation), like those grown with FOB (data not shown). However, when these cells were disrupted with glass beads, the protoporphyrinogen oxidase activity measured in vitro on cell extracts was completely inhibited in cells grown with FOB-AF (Figure 9D). Thus, protoporphyrinogen oxidase activity was not inhibited in vivo in cells that had accumulated enough siderophore drug conjugate to inhibit the enzyme fully in vitro, despite considerable dilution of the inhibitor (by about 1:1000) because of cell disruption in suspension buffer. The most probable explanation is that the drug was strictly compartmentalized in vivo, such that it could not reach its mitochondrial target unless the cells were completely disrupted. This observation, together with the fluorescence microscopy observations obtained with FOB-NBD, strongly supports the hypothesis that FOB is tightly compartmentalized in the vacuole after its uptake by the cells. It also shows the intrinsic limitation of coupling a drug to a siderophore to generate new antifungal compounds. In this case, FOB-AF cannot be used as an antifungal agent against S. cerevisiae, contrary to our expectations.


Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates.

Froissard M, Belgareh-Touzé N, Dias M, Buisson N, Camadro JM, Haguenauer-Tsapis R, Lesuisse E - Traffic (2007)

The AF-FOB conjugate does not reach its target in vivo.A) The IC50 values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. 55Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of 55Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.
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Related In: Results  -  Collection

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fig09: The AF-FOB conjugate does not reach its target in vivo.A) The IC50 values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. 55Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of 55Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.
Mentions: Acifluorfen is a powerful inhibitor of protoporphyrinogen oxidase, a mitochondrial enzyme catalysing the formation of protoporphyrin IX from protoporphyrinogen in the heme biosynthetic pathway. We covalently coupled this inhibitor to DFOB, using the same chemical coupling procedure as for Ga-DFOB-NBD (Methods) and assessed the inhibitory effect of (D)FOB-AF with respect to AF on protoporphyrinogen oxidase activity in vitro. The siderophore drug conjugate displayed weaker inhibitory activity than AF alone (Figure 9A), particularly when iron was bound to the siderophore moiety. This was probably because of steric constraints, as the substrate-binding domain (which is also the inhibitor-binding domain) of protoporphyrinogen oxidase encompasses a narrow active site cavity (37). Despite this limitation, the IC50 values measured with DFOB-AF and FOB-AF were nonetheless low enough (about 10−7 m and 10−6 m, respectively; Figure 9A) for the drug conjugates to be used as potent inhibitors of heme synthesis in yeast. Nevertheless, although AF-methyl efficiently inhibited S. cerevisiae growth on agar plates, the siderophore drug conjugate did not (Figure 9B). We then checked whether the cells took up FOB-AF efficiently. Both the fluorescent analog of FOB (FOB-NBD) and the siderophore drug conjugate (FOB-AF) were taken up by the cells through Sit1, although less efficiently than FOB itself (Figure 9C). We cultured cells bearing the SIT1 gene under control of the GAL1 promoter with galactose as the carbon source and in the presence of FOB-AF or FOB (100 μm). The washed pellets of cells cultured overnight in these conditions were brownish yellow in colour (the colour of undissociated siderophore), indicating that they had accumulated large amounts of FOB/FOB-AF (data not shown). The cells grown with FOB-AF showed no growth defect (and no porphyrin accumulation), like those grown with FOB (data not shown). However, when these cells were disrupted with glass beads, the protoporphyrinogen oxidase activity measured in vitro on cell extracts was completely inhibited in cells grown with FOB-AF (Figure 9D). Thus, protoporphyrinogen oxidase activity was not inhibited in vivo in cells that had accumulated enough siderophore drug conjugate to inhibit the enzyme fully in vitro, despite considerable dilution of the inhibitor (by about 1:1000) because of cell disruption in suspension buffer. The most probable explanation is that the drug was strictly compartmentalized in vivo, such that it could not reach its mitochondrial target unless the cells were completely disrupted. This observation, together with the fluorescence microscopy observations obtained with FOB-NBD, strongly supports the hypothesis that FOB is tightly compartmentalized in the vacuole after its uptake by the cells. It also shows the intrinsic limitation of coupling a drug to a siderophore to generate new antifungal compounds. In this case, FOB-AF cannot be used as an antifungal agent against S. cerevisiae, contrary to our expectations.

Bottom Line: Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells.Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments.Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Trafic intracellulaire des protéines dans la levure, Département de biologie Cellulaire, Institut Jacques Monod, Unité Mixte de Recherche 7592 CNRS-Universités Paris 6 et 7, France.

ABSTRACT
We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

Show MeSH
Related in: MedlinePlus